RODRIGO ALEX ARTHUR
“AVALIAÇÃO DE CARACTERÍSTICAS FENOTÍPICAS
DE CARIOGENICIDADE DE GENÓTIPOS DE
STREPTOCOCCUS MUTANS
ISOLADOS
DE BIOFILME DENTAL”
Tese apresentada à Faculdade de Odontologia de
Piracicaba, da Universidade Estadual de Campinas,
para obtenção de título de Doutor em Odontologia,
área de concentração em Cariologia.
Orientadora: Profa. Dra. Cínthia Pereira Machado Tabchoury Co-orientadora: Profa. Dra. Renata de Oliveira Mattos-Graner
Piracicaba
2010
FICHA CATALOGRÁFICA ELABORADA PELA
BIBLIOTECA DA FACULDADE DE ODONTOLOGIA DE PIRACICABA Bibliotecária: Marilene Girello – CRB-8a. / 6159
Ar77a Arthur, Rodrigo Alex. Avaliação de características fenotípicas de cariogenicidade de genótipos de streptococcus mutans isolados de biofilme dental. / Rodrigo Alex Arthur. -- Piracicaba, SP: [s.n.], 2010.
Orientadores: Cínthia Pereira Machado Tabchoury, Renata de Oliveira Mattos-Graner.
Tese (Doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
1. Placa dentária. 2. Virulência. 3. Microbiologia. I. Tabchoury, Cínthia Pereira Machado. II. Mattos-Graner, Renata de Oliveira. III. Universidade Estadual de Campinas. Faculdade de
Odontologia de Piracicaba. IV. Título. (mg/fop)
Título em Inglês: Evaluation of phenotypic traits of cariogenicity of Streptococcus
mutans genotypes isolated from dental biofilm
Palavras-chave em Inglês (Keywords): 1. Dental plaque. 2. Virulence. 3. Microbiology
Área de Concentração: Cariologia Titulação: Doutor em Odontologia
Banca Examinadora: Cínthia Pereira Machado Tabchoury, Denise Madalena Palomari Spolidorio, Cristiane Yumi Koga Ito, Adriana Franco Paes Leme, Marinês Nobre dos Santos Uchôa
Data da Defesa: 23-02-2010
DEDICATÓRIA
A Deus, pela presença constante, por iluminar e orientar meu caminho, e por permitir que mais uma conquista fosse alcançada.
Aos meus pais, Wilson e Sônia, pelo imenso carinho e amor. Pela simplicidade, paciência e pela sabedoria e pelo incentivo. Mesmo diante de dificuldades, sempre
fizeram o possível para que eu realizasse meus sonhos. Vocês são os co-autores de todas as minhas conquistas. Obrigado por permitirem que eu chegasse até
aqui.
Às minhas irmãs, Kelly e Karen, pela divertida convivência, pela alegria, pela amizade e pela confidência. Vocês tornam a minha vida muito mais feliz. Aos meus tios Fátima e Adilson, que sempre torceram por mim e vibraram com cada conquista. Por todo apoio, carinho e conselhos. Além de tios, vocês sempre
serão meus pais do coração.
Aos meus avós, José e Santina, pelos ensinamentos, pelas orações e pela sabedoria.
Aos meus tios, Ivete e César, Luzia e Aldrovando e João e Maria, e a todos meus familiares, pela presença e por todo apoio que sempre me deram. À Thais, pelo carinho imensurável. Pelo amor sincero e puro e por me mostrar o
valor das pequenas coisas. Pela paz de espírito, pelo apoio, por compartilhar sonhos e planos e por todos os momentos que temos passado juntos. Obrigado
por fazer parte da minha vida.
À família da Thais, que sempre me recebe e me acolhe com muito carinho. Com carinho, dedico.
AGRADECIMENTOS ESPECIAIS
À minha orientadora, Profa. Dra. Cínthia Pereira Machado Tabchoury, pela confiança e oportunidades oferecidas. Pela amizade construída desde minha Iniciação Científica e Mestrado, períodos em que também estive sob sua orientação. Pelo constante incentivo e pela inestimável contribuição à minha vida profissional. Obrigado por todo apoio, pela atenção e pela dedicação durante todos esses anos.
À minha co-orientadora, Profa. Dra. Renata de Oliveira Mattos-Graner, pela ajuda e pela competência. Pelas orientações e sugestões que me guiaram no decorrer da realização desse trabalho.
AGRADECIMENTOS ESPECIAIS
A Profa. Dra. Altair Antoninha Del Bel Cury, pela dedicação, pela determinação e pelas sugestões. Por toda preocupação e atenção dispensada aos alunos.
Ao Prof. Dr. Jaime Aparecido Cury, por ter contribuído para minha formação intelectual e científica desde a graduação. Por servir como exemplo de competência e dedicação. Obrigado pelos ensinamentos, pelo incentivo, pelas sugestões e pela amizade.
Ao Prof. Dr. Pedro Luiz Rosalen, pelo agradável convívio, pela disponibilidade, pela valiosa contribuição e ajuda durante o planejamento e execução desse trabalho.
Ao Prof. Dr. Bernhard Guggenheim, meu orientador durante os doze meses em que realizei estágio de doutorado no exterior (PDEE-CAPES) no Instituto de Microbiologia e Imunologia Oral (OMI) do Centro de Medicina Oral e Cirurgia Crânio-Maxilo-Facial (ZZMK), da Universidade de Zurique (Suíça). Agradeço pela atenção, cuidado, preocupação e pela forma amigável com que fui recebido em seu laboratório. Obrigado pela confiança, pela oportunidade e também pela valiosa contribuição para a minha formação profissional.
AGRADECIMENTOS
À Universidade Estadual de Campinas (UNICAMP), na pessoa de seu Magnífico Reitor, Prof. Fernando Ferreira da Costa.
À Faculdade de Odontologia de Piracicaba, na pessoa de seu Diretor, Prof.
Dr. Francisco Haiter Neto, por proporcionar a realização dessa pesquisa.
Ao Prof. Dr. Jacks Jorge Júnior, Coordenador Geral da Pós-Graduação da FOP-UNICAMP.
À Profa. Dra. Maria Beatriz Duarte Gavião, Coordenadora do Programa de Pós-Graduação em Odontologia.
À Profa. Dra. Livia Maria Andaló Tenuta, pela competência profissional, pelas sugestões e pelo convívio.
Aos professores do Programa de Pós-Graduação em Odontologia da FOP-UNICAMP, pelo constante aprendizado.
Aos professores Drs. Francisco Humberto Nociti Jr., Iriana Carla Zanin,
Jaime Aparecido Cury, Livia Andaló Tenuta e Pedro Luiz Rosalen pelas
sugestões e considerações feitas nos exames de Qualificação de primeira e de segunda fase.
À Érica Alessandra Pinho Sinhoreti e Raquel Q. Marcondes Cesar
Sacchi, secretárias da Comissão de Pós-Graduação; Maria Elisa dos Santos,
secretária do Programa de Pós-Graduação em Odontologia e à Eliete Riguetto, secretária do Departamento de Ciências Fisiológicas, pela atenção, dedicação e ajuda.
Aos técnicos, José Alfredo da Silva e Waldomiro Vieira, do Laboratório de Bioquímica Oral, e Eliane Melo Franco, do Laboratório de Farmacologia, da FOP-UNICAMP, pela amizade construída durante todos esses anos e por sempre estarem dispostos a ajudar.
Ao Ramiro Mendonça Murata e à Marlise Klein por me ensinarem as metodologias para análise fenotípica dos genótipos.
Aos alunos de Iniciação Científica, Lenita Marangoni Lopes, Maria Clara
Sayuri de Sousa, Nanna Mabele dos Santos, Patrícia Ribeiro Batista e Waldemir Vieira Junior, pelo agradável convívio. Aprendi muito trabalhando com
vocês.
Aos amigos Gláuber Campos Vale e Renzo Ccahuana-Vásquez, pela amizade e pela rica convivência durante esses seis anos de pós-graduação.
Aos amigos da Cariologia: Adelsilene Veras, Ana Flávia Calvo, Carolina
Aires, Danilo Catani, Gisele Moi, Juliana Braga, Karla Cook, Marília Correia, Paulo Peres, Regiane Amaral, Renata Cerezetti, Rosana Hoffmann, Sandro Kusano, pelo agradável convívio durante a pós-graduação.
Aos amigos Anna Maria Papa, Carolina Nóbrega, Claudia Zamataro,
Cristiane Duque, Karla Mychellyne, Maximiliano Cenci, Stela Pereira, Tatiana Pereira-Cenci, pela amizade preciosa.
Aos amigos da Farmacologia, da Microbiologia e da Prótese Parcial Removível, pelo companheirismo e pelo agradável convívio.
Aos profs. Drs. Rudolf Gmür e Jan van der Ploeg, do Instituto de Microbiologia e Imunologia Oral, da Universidade de Zurique, pelo convívio e por toda ajuda a mim oferecidas.
Aos técnicos, Andre Meier, Bärbel Sauer, Helga Lüthi-Schaller, Martin
Gander, Verena Osterwalder e Verena Böhm, e também à aluna Danusia Banu,
pela atenção, pela amizade e pela disponibilidade em me ensinar coisas novas durante meu estágio PDEE.
Ao Dr. Thomas Thurnheer¸ pela amizade e pelo agradável convívio durante minha estadia em Zurique.
À minha família em Zurique, Kelly e Andy Meier, Iashure e Reto, Irlane e
Mirko, Juliana e Bruno Jufer, Silvia e Thomas Thurnheer, pela preocupação,
pela atenção, pelos momentos de descontração, por todo apoio e pela amizade. Aos voluntários que gentilmente participaram dessa pesquisa. Sem eles, essa pesquisa não poderia ser realizada.
À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) e à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela concessão de bolsas (proc. nº 2005/03089-6 e BEX.1112/08-2) e pelo auxílio pesquisa (proc. nº 2007/08000-9).
A todos que direta ou indiretamente contribuíram para a realização desse estudo.
RESUMO
Os estreptococos do grupo mutans, em especial Streptococcus mutans, são considerados como um dos principais microrganismos relacionados à doença cárie dental. Clinicamente, esses microrganismos estão presentes na cavidade bucal na forma de diferentes genótipos, que podem apresentar diferentes características fenotípicas. Nesse sentido, foi avaliada diversidade genotípica de
S. mutans em biofilme dental formado in vivo ou in situ durante 3 dias sob
condições controladas de exposição à sacarose, o mais cariogênico dos carboidratos, e os seus monossacarídeos constituintes (glicose e frutose). Diferentes genótipos de S. mutans foram encontrados nessas distintas condições, entretanto, não foi observada seleção de genótipos nos biofilmes dentais formados. Além disso, as características fenotípicas de virulência desses genótipos não foram avaliadas. Sendo assim, seria importante avaliar nesses genótipos previamente isolados de biofilme dental formado in vivo e in situ, na presença ou ausência de sacarose, as características fenotípicas de cariogenicidade relacionadas à aciduricidade e à acidogenicidade, visando investigar a relação entre um alto desafio cariogênico (exposição frequente à sacarose e acúmulo de biofilme) e a virulência de S. mutans. Tanto naqueles genótipos previamente isolados de biofilme dental formado in vivo, quanto naqueles isolados de biofilme dental formado in situ, a aciduricidade foi avaliada em relação à análise da viabilidade celular em condições ácidas e em relação à atividade da bomba F-ATPase, e a acidogenicidade foi avaliada em relação à análise da curva de queda de pH devido metabolização de glicose. Além disso, oito genótipos previamente isolados do biofilme dental formado in situ durante 3 dias foram submetidos a um crescimento na forma de biofilme in vitro, condição na qual foram avaliadas a acidogenicidade, a habilidade do genótipos em sintetizar polissacarídeos extracelulares e o potencial desses genótipos para desmineralizar o esmalte dental. Em relação aos genótipos isolados de biofilme dental formado in
vivo, aqueles isolados de biofilme formado na presença de sacarose foram mais
apresentaram menores valores de pH final durante a análise da curva de queda de pH e também maior velocidade na produção de ácidos nos primeiros 15 minutos de metabolização da glicose, que aqueles encontrados em biofilme formado na sua ausência de sacarose. Além disso, não foram encontradas diferenças expressivas na atividade da bomba F-ATPase entre essas duas condições distintas. Comportamento semelhante também foi observado para genótipos previamente isolados de biofilme dental formado in situ, com maior aciduricidade e acidogenicidade para aqueles genótipos isolados de biofilme formado na presença de sacarose. No modelo de biofilme in vitro não foram encontradas diferenças nem na acidogenicidade nem na habilidade dos genótipos de produzirem polissacarídeos extracelulares. Entretanto, os genótipos apresentaram potenciais cariogênicos distintos, não havendo relação entre o potencial cariogênico desses genótipos e a condição na qual esses genótipos foram isolados (presença ou ausência de um alto desafio cariogênico). Os resultados sugerem que as freqüentes quedas de pH decorrentes da exposição à sacarose parecem tornar os genótipos mais de S. mutans mais virulentos. Além disso, genótipos distintos de S.
mutans podem apresentar diferentes potenciais cariogênicos.
Palavras-chave: Streptococcus mutans, genótipos, sacarose, aciduricidade,
ABSTRACT
Mutans streptococci, mainly Streptococcus mutans, are considered as the main microorganisms related to dental caries. These microorganisms are present in oral cavity as distinct genotypes, which may show distinct phenotypic traits. In this context, it was evaluated the S. mutans genotypic diversity in dental biofilm formed in vivo and in situ during 3 days under controlled exposure to sucrose, the most carigenic carbohydrate, and its monosaccharides constituents (glucose and fructose). Distinct S. mutans genotypes were found under these before mentioned conditions, but no selection of them was found in dental biofilms formed. Moreover, the virulence phenotypic traits of these genotypes were not evaluated. Thus, it would be important to evaluate in these genotypes previously isolated from dental biofilm formed in vivo and in situ in the presence or absence of sucrose, the phenotypic traits of cariogenicity related to aciduricity and acidogenicity in order to investigate the relationship between a high cariogenic challenge (frequent exposure to sucrose and biofilm accumulation) and their virulence. Either in those genotypes isolated from in vivo dental biofilms, or in those isolated from in situ dental biofilms, the acidogenicity trait was evaluated through counts of viable cells in acid conditions and F-ATPase activity, and the acidogenicity trait was evaluated through the ability to lower the pH due to glycolysis. Besides, eight genotypes previously isolated from in situ dental biofilms formed during three days were grown as in vitro biofilms, and these genotypes were evaluated regarding their acidogenicity, cariogenic potential and ability to synthesize extracellular polysaccharides. In relation to genotypes isolated from in vivo biofilms, those isolated from biofilms formed in the presence of sucrose were more acid-tolerant, either at pH 5.0 or at pH 2.8, and more acidogenic, since they showed lower values of final pH during the evaluation of the curve of pH fall and also higher ability to produce acids in the first 15 minutes of glucose fermentation, than genotypes isolated from in vivo biofilms formed in the absence of sucrose. Besides, no expressive differences regarding F-ATPase activity between these two distinct conditions were found. Genotypes found only in the presence of sucrose were
more acidogenic than those found only in the absence of this carbohydrate. Similar data were found for genotypes isolated from in situ dental biofilms formed in the presence of sucrose, which were more aciduric and more acidogenic than genotypes isolated from biofilms formed in the absence of this carbohydrate. In in
vitro biofilm model, no differences either in the acidogenicity or in the ability of
genotypes to synthesize extracellular polyssacharide were found. However, the S.
mutans genotypes showed distinct cariogenic potential, independent of the fact
that these genotypes had been isolated in the presence or absence of a high cariogenic challenge. The results suggest that frequent pH fall due to sucrose exposure may select more virulent S. mutans genotypes. Besides, distinct S.
mutans genotypes may show distinct cariogenic potential.
Key-words: Streptococcus mutans, genotypes, sucrose, aciduriciry, acidogenicity,
SUMÁRIO
INTRODUÇÃO GERAL……….1
CAPÍTULO 1: “Genotypic and phenotypic analysis of S. mutans isolated from dental biofilm formed in vivo under a high cariogenic condition”………..……..6
CAPÍTULO 2: “Phenotypic analysis of S. mutans genotypes in planktonic and biofilm conditions”……….28
DISCUSSÃO GERAL………...54
CONCLUSÃO GERAL………...57
REFERÊNCIAS SUPLEMENTARES………...58
APÊNDICE...67
ANEXO 1: Certificado de aprovação pelo Comitê de ética em Pesquisa...74
ANEXO 2: Comprovante de submissão de artigo científico para publicação...76
ANEXO 3: Declaração de não infração dos direitos autorais...77
INTRODUÇÃO GERAL
A cárie dental é uma doença biofilme-açúcar dependente relacionada à frequente ingestão de carboidratos fermentáveis (Bowen et al., 1980). Dentre os carboidratos da dieta, a sacarose é considerada como o mais cariogênico (Paes Leme et al., 2006), pois além de ser fermentada a ácidos, reduzindo o pH do biofilme dental, é substrato para a síntese de polissacarídeos extracelulares (PECs) (Rölla et al., 1985; Cury et al., 2000). Esses PECs, principalmente os insolúveis, tornam o biofilme mais poroso (Dibdin & Shellis, 1988), facilitando a difusão de ácidos (Zero et al., 1986; 1992), que são capazes de provocar desmineralização em esmalte dental (Cury et al., 2000; Pecharki et al., 2005; Ccahuana-Vásquez et al., 2007; Vale et al., 2007) e em dentina (Aires et al., 2002, 2008). Além disso, essas quedas de pH no biofilme dental são consideradas como um dos principais estresses aos quais os microrganismos orais estão expostos (Lemos et al., 2005), podendo alterar a homeostasia microbiana do biofilme, influenciando o crescimento e a sobrevivência dos microrganismos orais (Bowden & Hamilton, 1998; Marsh, 2003).
Nesse contexto, os estreptococos do grupo mutans, em especial o
Streptococcus mutans, têm sido considerados como um dos principais
microrganismos relacionados à cárie dental (Löesche, 1986; Tanzer et al., 2001). Isso se deve ao fato desses microrganismos apresentarem algumas características de virulência, tais como, a aciduricidade, que é a capacidade de sobreviverem e crescerem nos ambientes e nos períodos de reduzido pH decorrentes da metabolização dos carboidratos, e a acidogenicidade, que é a capacidade de produzirem ácidos mesmo em condições de baixo pH (Banas, 2004). Essas características conferem vantagem adaptativa aos S. mutans em relação aos outros microrganismos do biofilme em períodos de acidificação do meio. Além dessas características de virulência, os S. mutans também apresentam a particularidade de sintetizarem PECs, por ação enzimática de glicosiltransferases, utilizando sacarose como substrato (Hamada & Slade, 1980;
Bowen et al., 2002), que viabilizam a aderência desses microrganismos à superfície dental (Rölla, 1989).
Sabe-se que os ácidos produzidos pelos S. mutans devido à metabolização de carboidratos fermentáveis poderiam acarretar a acidificação do meio intracelular, comprometendo o funcionamento celular, principalmente das enzimas ácido-sensíveis. Para evitar o comprometimento da viabilidade celular, os microrganismos possuem um sistema, denominado “bomba translocadora de prótons” (F-ATPase) localizada na membrana celular, que bombeia os íons hidrogênio para o meio extracelular. Dessa forma, dentre os diversos mecanismos relacionados à aciduricidade dos S. mutans, o sistema F-ATPase tem sido considerado como um dos mais importantes (Lemos et al., 2005). Como esse transporte é contrário ao gradiente de difusão desses íons, há gasto de energia, que é fornecida pela hidrólise de moléculas de ATP (Deckers-Hebestreit & Altendorf, 1996). Nesse contexto, alguns estudos têm demonstrado o importante papel desempenhado pela bomba F-ATPase na manutenção da viabilidade celular dos S. mutans (Belli & Marquis, 1991; Hamilton & Buckley, 1991; Nascimento et
al., 2004a).
Clinicamente, alguns trabalhos têm demonstrado que existem vários genótipos de S. mutans, tanto na saliva quanto no biofilme dental (Caufield & Walker, 1989; Alaluusua et al., 1996; Saarela et al., 1996; Mattos-Graner et al., 2001; Lindquist & Emilsson, 2004; Klein et al., 2004; Napimoga et al., 2004; Nascimento et al., 2004b; Nogueira et al., 2005; Cogulu et al., 2006; Gou et al., 2006; Lembo et al., 2007; Tabchoury et al., 2008; Alves et al., 2009). Em média, de 1 a 5 genótipos distintos de S. mutans têm sido identificados por indivíduo (Saarela et al., 1993; 1996; Gronroos & Alaluusua et al., 2000; Redmo-Emanuelsson & Thornquist 2000, 2001; Redmo-Redmo-Emanuelsson et al., 2003; Klein et
al., 2004; Lemos et al., 2007; Tabchoury et al., 2008), sendo que genótipos
distintos podem colonizar uma mesma superfície dental, e que um mesmo genótipo também pode colonizar diferentes sítios (Redmo-Emanuelsson et al., 2003). Esses diferentes genótipos, principalmente os mais prevalentes, foram
ainda detectados 4, 7, 18, 20, 24, 36, 60 e 84 meses após a genotipagem inicial (Redmo-Emanuelsson & Thornquist 2000, 2001; Redmo-Emanuelsson et al., 2003; Klein et al., 2004; Lindquist & Emilson, 2004; Alves et al., 2009), sugerindo que os mesmos são estáveis na cavidade bucal. Interessantemente, não tem sido encontrada qualquer correlação entre níveis de S. mutans na saliva e diversidade genotípica (Gronroos & Alaluusua, 2000; Mattos-Graner et al., 2001; Lembo et al., 2007; Alves et al., 2009) e nem entre diversidade genotípica de S. mutans e atividade de cárie (Gronroos & Alaluusua et al., 2000; Redmo-Emanuelsson & Thornquist, 2000; Mattos-Graner et al., 2001; Lemos et al., 2007). Porém, alguns trabalhos têm sugerido que indivíduos cárie-ativos possuem menor diversidade genotípica de S. mutans (Kreulen et al., 1997; Redmo-Emanuelsson et al., 2003), enquanto que outros trabalhos têm sugerido o oposto (Alaluusua et al., 1996; Napimoga et al., 2004; Alves et al., 2009).
Em relação a essa diversidade genotípica presente na cavidade bucal, tem sido demonstrado que genótipos distintos podem apresentar diferentes capacidades de virulência. Mattos-Graner et al. (2004) verificaram que diferentes genótipos de S. mutans apresentam diferentes atividades de glicosiltransferase. Guo et al. (2006) mostraram que diferentes genótipos de S. mutans apresentaram diferença na síntese de polissacarídeo extracelular insolúvel, na acidogenicidade e na aciduricidade. Além disso, Napimoga et al. (2004) observaram que genótipos de S. mutans isolados de sítios com cárie ativa apresentavam maior habilidade para sintetizar polissacarídeo extracelular insolúvel quando comparados àqueles genótipos presentes em sítios livres de cárie. Maior aciduricidade também foi encontrada em genótipos de crianças cárie-ativas quando comparada aos genótipos de crianças livres de cárie (Lembo et al., 2007).
De uma forma geral, essas características fenotípicas têm sido avaliadas em S. mutans cultivados como células planctônicas, porém pouco se sabe sobre o potencial cariogênico desses genótipos quando cultivados na forma de biofilmes. Nesse contexto, tem sido sugerido que o comportamento de microrganismos aderidos a superfícies pode ser diferente quando comparado ao comportamento
como célula planctônica. Alguns trabalhos têm demonstrado que a expressão protéica e gênica de S. mutans em biofilmes é diferente em relação à expressão em culturas planctônicas (Svensäter et al., 2001; Marsh, 2004; Shemesh et al., 2007). Além disso, tem sido demonstrado que S. mutans em biofilmes são mais acidúricos em relação a células planctônicas (Li et al., 2001; Welin-Neilands & Svensäter, 2007), o que demonstra que podem ocorrer modificações no fenótipo dos microrganismos por ocasião do crescimento em biofilmes.
No contexto dos estudos de diversidade genotípica na cavidade bucal, Arthur et al. (2006; 2007) avaliaram a diversidade de S. mutans isolados de um biofilme dental formado in vivo e in situ na presença de sacarose (indutor da síntese de PEC e carboidrato fermentável a ácido, promovendo queda de pH no biofilme) e de seus monossacarídeos constituintes, glicose e frutose (somente fermentáveis a ácido). Foi encontrada diversidade genotípica no biofilme dental entre os voluntários, entretanto, não foi verificada seleção de genótipos devido ao estresse induzido pelo metabolismo da sacarose nem pela fermentação de seus monossacarídeos constituintes. Genótipos específicos foram encontrados em menores proporções em cada uma dessas condições descritas, entretanto não foram encontradas diferenças expressivas na diversidade genotípica entre biofilmes formados sob diferentes desafios cariogênicos. Porém, as características fenotípicas de cariogenicidade dos genótipos isolados mediante esses diferentes desafios cariogênicos não foram exploradas. Sendo assim, seria importante avaliar as características fenotípicas de aciduricidade e de acidogenicidade dos genótipos isolados em ambos os biofilmes (in vivo e in situ), visando investigar a relação entre um alto desafio cariogênico (exposição frequente à sacarose e acúmulo de biofilme) e a virulência de S. mutans.
Dessa forma, o objetivo do primeiro trabalho (Capítulo 1) foi avaliar as características fenotípicas de cariogenicidade, tais como aciduricidade, por meio da análise de viabilidade celular em condições ácidas e da atividade da bomba F-ATPase, e a acidogenicidade, por meio de análise da habilidade em reduzir o pH devido à glicólise, de genótipos de S. mutans previamente isolados de biofilme
dental formado in vivo sob um alto desafio cariogênico (Arthur et al., 2006). O objetivo do segundo trabalho (Capítulo 2) foi avaliar essas mesmas características de cariogenicidade, relacionadas à aciduricidade e à acidogenicidade, em células planctônicas de genótipos de S. mutans previamente isolados de biofilme in situ formado durante 3 dias sob um alto desafio cariogênico (Arthur et al., 2007) e também a habilidade de sintetizar polissacarídeos extracelulares e o potencial de desmineralizar o esmalte dental de alguns genótipos num modelo de biofilme in
CAPÍTULO 1
Journal section for manuscript publication: Microbial ecology
Genotypic and phenotypic analysis of S. mutans isolated from dental biofilm formed in vivo under a high cariogenic condition
Rodrigo A. Arthur1, Altair A. Del Bel Cury2, Renata O. Mattos-Graner3, Pedro L.
Rosalen1, Gláuber C. Vale1, Jaime A. Cury1, Cínthia P.M. Tabchoury1*
Departments of 1Physiological Sciences, 2Prosthodontics and Periodontology and
3Oral Diagnosis from Piracicaba Dental School, UNICAMP, São Paulo, Brazil.
*corresponding author
Mailing address: Piracicaba Dental School Av. Limeira, 901 CEP 13414-903 Piracicaba, SP, Brazil Phone: #55-19-21065304 Fax: #55-19-21065212 E-mail: cinthia@fop.unicamp.br
RUNNING TITLE: S. mutans genotypes and phenotypic traits
This work was based on a thesis submitted by the first author to the Faculty of Dentistry of Piracicaba, University of Campinas, SP, Brazil, in partial fulfillment of the requirements for the Doctor Degree in Dentistry (Cariology Area)
Esse manuscrito foi submetido para publicação ao periódico
ABSTRACT
Oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of genotypes isolated under in vivo controlled conditions of high cariogenic challenge. This study aimed to evaluate the genotypic diversity of S.
mutans isolated from dental biofilms formed in vivo under frequent exposure or not
to sucrose, and the aciduricity and acidogenicity of these genotypes. Volunteers rinsed with distilled deionized water or 20% sucrose solution for 10 sec, 8 x/day, during 3 days for biofilm formation on upper posterior teeth. S. mutans isolates collected from saliva and biofilms were analyzed for their genotypic identity by arbitrarily-primed PCR. Biofilm genotypes were evaluated regarding their acid susceptibility, F-ATPase activity and ability to lower the pH through glycolysis. Most of the volunteers harbored only one genotype in saliva, which was detected in almost all biofilm samples always at higher proportion, but other specific genotypes were also found in a lower proportion in the biofilms. Genotypes from biofilms exposed to sucrose showed higher acid tolerance at pH 5.0 and 2.8 after 60 and 30 min of incubation, respectively. Genotypes exclusively isolated from biofilms exposed to sucrose showed higher acidogenicity than those exclusively isolated from biofilms not exposed to this carbohydrate. Even though the results suggest that there were no expressive differences in genotypic diversity between biofilms formed under exposure or not to sucrose, it seems that biofilms formed under a high cariogenic condition harbored more virulent genotypes.
INTRODUCTION
Dental caries is a dietary and biofilm-dependent disease related to the frequent consumption of fermentable carbohydrates (5) and to the shift in biofilm microbiota induced by the pH fall (18). Among the dietary carbohydrates, sucrose is the most cariogenic because, besides being fermented, it is also the unique substrate for the synthesis of extracellular polysaccharide (EPS) (8, 28), which may improve bacterial adherence to tooth surfaces as well as modify the matrix of dental biofilm (25).
Among the cariogenic microorganisms colonizing the dental biofilm, the one most implicated in dental caries is mutans streptococci, especially Streptococcus
mutans (17). S. mutans produce EPS from sucrose and are acidogenic and
aciduric bacteria, that is, they metabolize fermentable carbohydrates, producing acids that decrease the biofilm pH, and also show the ability of surviving, growing and maintaining their metabolism in this acidic condition (18). This acid tolerance trait is partly due to the presence of a membrane-bound protein called F-ATPase, which extrudes protons out of the cells, preventing the intracellular pH fall and a consequent damage to acid-sensitive enzymes, DNA and proteins (26).
Several studies have shown that the oral cavity harbors distinct S. mutans genotypes, either in saliva or dental biofilms (15, 27, 29). Additionally, it has already been reported that genotypes could differ in their virulence abilities (20, 22), which may facilitate their ability to colonize and even predominate in a environment under a high cariogenic condition. Genotypic diversity was also observed among volunteers in in situ dental biofilms formed under sugar stress exposure (2), but no specific genotypes were selected due to the stress induced by sucrose metabolism or simple fermentation of its monosaccharides. However, little is known about the genotypic diversity of S. mutans and their phenotypic traits in a dental biofilm formed in vivo under a frequent and controlled exposure to sucrose.
Thus, this study aimed to evaluate the genotypic diversity of S. mutans isolates from in vivo dental biofilms, formed under high cariogenic challenge
(biofilm accumulation under frequent exposure to sucrose), as well as some virulence traits related to aciduricity and acidogenicity of the genotypes.
MATERIALS AND METHODS
Selection of volunteers and study design
This study was approved by the Research and Ethics Committee of
Piracicaba Dental School (protocols no. 053/2004 and 004/2006). A total of 12
healthy adults were screened for salivary levels of mutans streptococci (MS). From that group, a subset of 6 volunteers (18 to 28 years old) were selected, due to high
MS counts in saliva (≥ 105 colonies forming units/mL; CFU/mL) and ability to
comply with the experimental protocol. The exclusion criteria included antibiotic use for the last 2 months before starting the study, use of any form of medication that modifies salivary secretion, use of fixed or removable orthodontic appliance, periodontal disease or general/systemic illness. Biofilms formed in vivo in the absence or in the presence of sucrose on the surfaces of upper pre-molars and molars were collected from these subjects for the analysis of genotypic diversity of
S. mutans by arbitrarily-primed PCR (AP-PCR). Afterwards, all biofilm S. mutans
genotypes were phenotypically evaluated regarding their aciduricity, through evaluation of acid susceptibility and F-ATPase activity, and acidogenicity, by evaluation of the ability to lower the pH through glycolysis.
Saliva sampling and microbiological analysis
Stimulated whole saliva samples were collected from the individuals in the morning within fasting condition and without previous teeth brushing. Saliva was diluted in sterile 0.9% NaCl, and inoculated in triplicate onto Mitis Salivarius Agar (Difco, Sparks, MD, USA) plates supplemented with 20% sucrose (Merck, Darmstadt, Germany) and 0.2 U of bacitracin/mL (Sigma, Steinheim, Germany) (MSB), for culturing of mutans streptococci group. All plates were incubated at 37ºC for 48 h in an atmosphere of 10% CO2. Eight representative morphological types of S. mutans colonies were collected from MSB plates previously inoculated
with saliva samples, subcultured on mitis salivarius agar (MSA) and Brain Heart Infusion (BHI) agar (Difco, Sparks, MD, USA), and pure cultures stored at -70ºC in 10% skim milk medium (Difco, Sparks, MD, USA) for further genotypic analysis (2). The purity and identity of the isolates were checked by Gram’s stain and colonial morphology on MSA.
In vivo biofilm formation and biofilm collection
An in vivo cross-over and blind study was conducted in 2 experimental phases. During 3 days, the six selected volunteers rinsed with 15 mL of 20% sucrose solution or distilled deionised water, for 10 sec, 8 x/day, at predetermined times (13). The volunteers were randomly assigned to the above mentioned conditions of presence or absence of sucrose during biofilm formation and were instructed to neither brush the upper pre-molars and molars nor use dental floss in these teeth, during the experimental phases, for biofilm accumulation. A wash-out period of 15 days was carried out between both experimental phases. Distilled and deionized water and sucrose solutions were handed daily to the volunteers. At the end of each 3-day experimental phase, 10 h after the last exposure to the respective solution, biofilms formed on upper pre-molars and molars were collected with a sterile spatula, in the morning, with the volunteers in fasting conditions and without having brushed their teeth. The biofilm was weighed (± 0.01 mg), suspended in 0.9% NaCl sterile solution (1 mL/mg wet weight), sonicated (8) (Sonics and Materials, Danbury, CT, USA), serially diluted and inoculated in duplicate in MSB. All plates were incubated at 37ºC for 48 h at 10% CO2.
Isolation of S. mutans strains and extraction of genomic DNA
From seven to eight colonies, representing all morphological types of S.
mutans, were collected from cultures of saliva and in vivo biofilm samples on MSB
plates and pure cultures were frozen at -70ºC in 10% skim milk (2). For genomic DNA extraction, aliquots from saliva and biofilm samples were collected from skim milk and plated on BHI agar (Difco, Sparks, MD, USA), which was incubated at
37ºC for 24 h at 10% CO2. The colonies from BHI agar were inoculated into 3 mL of Todd Hewitt Broth (Difco, Sparks, MD, USA) and incubated at 37ºC for 18 h at 10% CO2. Cells from these cultures were then harvested and genomic DNA was extracted from the cell pellet (21). Integrity of the genomic DNA samples was checked at samples electrophoretically resolved in 1% agarose gel (Invitrogen, Spain) and stained with ethidium bromide (5 µg/mL). PCR reactions with species-specific primers to gtfB and gbpB genes were adopted in order to confirm the identity of the S. mutans isolates (23, 19).
Genotypic analysis
Arbitrarily primed PCR (AP-PCR) assays were performed with the arbitrary primer OPA 02 (5´-TGCCGAGCTG-3´) (16). The amplifications occurred under the following conditions: 95ºC for 2 min, for initial denaturation, and 45 cycles of 94ºC for 30 sec (denaturation), 36ºC for 30 sec (annealing) and 72ºC for 1 min (extension) and a final extension at 72ºC for 5 min. Genomic DNA of S. mutans strain UA 130 (kindly provided by Dr. Page W. Caufield, New York University, NY, USA) and distilled deionized water were applied in all PCR baths, as positive and negative controls, respectively. Products of AP-PCR were electrophoretically resolved in agarose gels that were run at 3 V/cm during 3 h in Tris-Borate-EDTA (TBE) running buffer. Gels were stained with ethidium bromide solution (5 µg/mL) (Invitrogen, Carlsbad, CA, USA) for 10 min and their images captured by a digital imaging system (Gel logic 100 Imaging System, Kodak, Japan).
For analysis of the S. mutans genotypic profiles from the same volunteer, AP-PCR products from the isolates obtained from saliva and dental biofilms were always resolved side-by-side in the same gel for visual comparisons (2). Thus, samples representative of each genotype were re-run side-by-side in a subsequent gel for direct comparisons of genotypes identified within distinct saliva or biofilms samples. The genotypes found were descriptively analyzed and their proportion, in relation to the number of colonies isolated in each sample and condition, was calculated.
Phenotypic analysis
For the phenotypic analysis, all genotypes identified in dental biofilm were reactivated from frozen stocks on BHI agar plates, which were incubated at 37ºC for 48 h at 10% CO2. Thus, CFU were transferred to tubes containing BHI broth, which were then incubated at 37ºC for 18 h at 10% CO2.
The ability of genotypes to withstand acid challenge was evaluated by acid killing assay (15). Briefly, aliquots of the 18-h growth BHI broth were transferred to tubes containing fresh BHI broth medium and grown until mid-exponential phase (OD550 = 0.5). Then, the suspension was centrifuged and the pellet was washed once with 0.1 M glycine buffer (pH 7.0) (Fluka, Steinheim, Germany). In addition, the washed pellets were resuspended in 0.1 M glycine buffer pH 7.0 (control) and at pH 5.0 or 2.8. Immediately after the resuspension (T0), after 30 (T30) and 60 min (T60) of incubation at 37ºC, aliquots were serially diluted, plated on BHI agar plates and incubated at 37ºC for 48 h at 10% CO2. Cell viability at each time was expressed as the ratio between counts of viable cells at pH 5.0 or 2.8 in relation to counts of viable cells at pH 7.0 at each time.
For F-ATPase assay, aliquots of the 18-h growth BHI broth were centrifuged and resuspended in 75 mM Tris-HCl plus 10 mM MgSO4. Then, the cells were permeabilized both with toluene (Merck, Darmstadt, Germany) and by freezing and heating cycles. The permeabilized cells were incubated with 0.5 M adenosine 5´-triphosphate (ATP) (Sigma, Steinheim, Germany) during 10 min in 50 mM Tris-maleate buffer (pH 6.0) (Sigma, Japan) with 10 mM MgSO4 (Fluka, Steinheim, Germany) (3). Inorganic phosphorous released from ATP was determined by the method of Bencini et al. (4). ATPase activity of each genotype was expressed as micromoles of phosphate released from ATP per gram of dry cell per minute of reaction. The standard unit of ATPase activity is 1 µmol of phosphate released per min (3).
In addition, the ability of S. mutans genotypes to lower the pH through glycolysis was monitored (3). Aliquots of the 18-h growth BHI broth were
centrifuged and resuspended in 50 mM KCl plus 1 mM MgCl2 solution (Fluka, Steinheim, Germany). The pH of the solution was adjusted to 7.2 and glucose was added in a final concentration of 55.6 mM. Then, the pH drop was assessed during 180 min with glass electrode, previously calibrated with pH standards (pH 4.0 and 7.0). The area under the curve of pH fall after 180 min (AUC) was calculated (considering pH 3.0 as a cut-off point). Also, the pH data were converted into
hidrogenionic concentration (cH+) and the cH+ area either between time zero and
after 15 min or between time zero and 180 min was calculated with the pH Plaque®
software (14). The acidogenicity was expressed as AUC and final pH (after 180
min) and cH+ area (after 15 and 180 min). All the assays described above were
conducted in duplicate in 3 distinct experiments, the genotypes were codified and S. mutans UA 159 was used as control.
Statistical analysis
For statistical analysis, the phenotypic traits of all genotypes isolated from biofilm formed in the absence of sucrose were compared to all genotypes isolated from biofilms formed in the presence of sucrose. Also, the phenotypic traits of genotypes exclusively isolated from biofilms formed in the absence of sucrose were compared to those genotypes isolated only from biofims formed in the presence of this carbohydrate. The assumption of equality of variances and normal distribution of errors were checked for all the response variables tested. Data that violated these assumptions were transformed when necessary (6) and submitted to t-test. When no transformation was adequate to normalize data (ratio between counts of viable cells at pH 2.8 T30 in relation to counts at pH 7.0 T30 for both
comparisons and AUC and cH+ area (after 180 min)for the second comparison),
the data were analyzed by Wilcoxon non-parametric test. SAS software system (version 8.02, SAS Institute Inc., Cary, NC, USA) was used, and the significance limit was set at 5%.
RESULTS
Distribution of S. mutans genotypes from saliva and in vivo biofilms
A total of 48 and 95 representative colonies of S. mutans were isolated from saliva and biofilms, respectively. All of the isolates were identified as S. mutans species, as determined in PCR reactions with species-specific primers for gtfB and
gpbB genes. A total of 19 distinct genotypes were identified in saliva and in in vivo
biofilm samples (Table 1). AP-PCR genotypic profiles from volunteer F are depicted in Figure 1. In 5 volunteers, only one S. mutans genotype was identified in the respective saliva samples, whereas in one volunteer (volunteer B) two genotypes were detected in saliva (Table 1). All genotypes identified in saliva samples were also detected in biofilm samples of the respective volunteer, independently of sucrose exposure. One exception was volunteer F, whose
salivary genotype was not found in the biofilm formed in the absence of sucrose exposure. In addition, besides the salivary genotypes, others were found in the biofilm formed either under sucrose exposure (4 genotypes: 7su, 10su, 11su and 13su) or not (8 genotypes: 4ddw, 6ddw, 9ddw, 16ddw, 17ddw, 18ddw and 19ddw) (Table 1). However, these genotypes were generally detected in lower proportions (12.5%) in biofilm when compared with genotypes also detected in saliva samples (Table 1).
Table 1. Genotypic diversity of S. mutans (%; percentage of each genotype in
relation to the total numbers of isolated colonies) in saliva and in vivo dental biofilm according to the presence or absence of sucrose during biofilm formation:
Distinct numbers represent different genotypes. sa: genotypes isolated from saliva;
ddw: genotypes isolated from biofilm formed in the absence of sucrose su: genotypes isolated from biofilm formed in the presence of sucrose.
Biofilm formation Volunteer Saliva Absence of
sucrose Presence of sucrose
A 1sa (100) 1ddw (100) 1su (100) B 2sa (85.7) 3sa (14.3) 2ddw (87.5) 4ddw (12.5) 2su (100) C 5sa (100) 5ddw (87.5) 6ddw (12.5) 5su (87.5) 7su (12.5) D 8sa (100) 8ddw (87.5) 9ddw (12.5) 10su (12.5) 8su (75) 11su (12.5) E 12sa (100) 12ddw (100) 12su (87.5) 13su (12.5) F 14sa (100) 15ddw (50) 16ddw (12.5) 17ddw (12.5) 18ddw (12.5) 19ddw (12.5) 14su (100)
Figure 1. AP-PCR fingerprinting profiles of representative strains of S. mutans
isolated from volunteer F from saliva and in vivo biofilms according to experimental treatments; 250-bp DNA ladder is showed in lane 1; lane 2 corresponds to genotype present in saliva; lanes 3 to 11 correspond to different isolates of S.
mutans according to the presence or absence of sucrose during biofilm formation;
positive control (S. mutans UA 130 – Forsyth Institute, Boston, Massachussets) and negative control (water) were present in lanes 12 and 13, respectively. Genotypes and condition of biofilm formation are represented at the bottom of the Figure. ddw: biofilms formed in the absence of sucrose; su: biofilms formed in the presence of sucrose. 250 1000 5000 1 2 3 4 5 6 7 8 9 10 11 12 13 14sa 15 16 17 15 18 19 14 14 14 ddw su 250 1000 5000 1 2 3 4 5 6 7 8 9 10 11 12 13 250 1000 5000 1 2 3 4 5 6 7 8 9 10 11 12 13 14sa 15 16 17 15 18 19 14 14 14 ddw su
Phenotypic Characteristics
All genotypes isolated from biofilms formed in the presence of sucrose showed significantly higher ratio of counts of viable cells at pH 2.8 (T30) in relation to counts of viable cells at pH 7.0 (T30) when compared to genotypes isolated from biofilms formed in the absence of sucrose (p<0.05) (Table 2). Also, statistically higher ratio of counts of viable cells at pH 5.0 (T60) in relation to counts of viable cells at pH 7.0 (T60) were also found for genotypes exclusively isolated from biofilms formed under sucrose exposure. In addition, a numerically higher activity of F-ATPase was observed for all genotypes isolated from biofilms formed in the presence of sucrose in comparison with those from biofilms formed in the absence of this carbohydrate, but the differences were not statistically significant (p=0.06) (Table 2).
With regard to the acidogenicity traits, genotypes exclusively found in biofilms formed under sucrose exposure (7su, 10su, 11su and 13su) showed
higher cH+ area (after 15 min) (p<0.01) and lower final pH (p<0.05) than those
found exclusively in biofilms not exposed to sucrose (4ddw, 6ddw, 9ddw, 15ddw, 16ddw, 17ddw, 18ddw and 19ddw) (Table 3).
Table 2. Cell viability at pH 5.0 and 2.8 (ratio in relation to cell viability at pH 7.0 at each time), and F-ATPase activity of the
genotypes isolated from in vivo biofilms (mean ± sd):
Means of each type of comparison followed by distinct letters differ statistically (p<0.05).
Means of each type of comparison, which are not followed by letters, do not differ from each other. §: data transformed by log10;
pH 5.0 pH 2.8
Comparison Condition of biofilm formation T0 T30 T60 T0 T30
(x 10-2) T60 F-ATPase activity (µmol/g/min) Absence of sucrose (n=13) 1.1 ± 0.3 1.1 ± 0.4 § 0.9 ± 0.3§ 1.0 ± 0.4 0.03 ± 0.09B 0.0 ± 0.0 16.1 ± 8.5 All genotypes Presence of sucrose (n=10) 1.1 ± 0.2 1.2 ± 0.6 § 1.0 ± 0.2 § 1.2 ± 0.6 0.1 ± 0.2A 0.0 ± 0.0 25.1 ± 13.8 Absence of sucrose (n=8) 1.0 ± 0.2 1.0 ± 0.4 § 0.8 ± 0.2B 0.9 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 15.9 ± 8.6 Exclusively-isolated
genotypes Presence of sucrose
(n=4) 1.2 ± 0.04 1.0 ± 0.2
Table 3. Acidogenicity traits of the genotypes isolated from in vivo biofilms (means ± sd):
Comparison Condition of biofilm
formation (after 180 min) AUC
(min*pH) Final pH (after 180 min) cH + area (after 15 min) (µmol/l/min) cH+ area (after 180 min) (µmol/l/min) Absence of sucrose (n=13) 315.0 ± 58.7 # 4.0 ± 0.1 0.3 ± 0.1§ 131.8 ± 58.7 All genotypes Presence of sucrose (n=10) 321.2 ± 105.5 # 3.9 ± 0.2 0.81 ± 1.3§ 147.2 ± 60.9 Absence of sucrose (n=8) 298.1 ± 17.0 3.9 ± 0.06 A 0.4 ± 0.1B § 139.5 ± 16.2 Exclusively-isolated
genotypes Presence of sucrose
(n=4) 295.0 ± 13.1 3.7 ± 0.1
B
(n=3)* 2.3 ± 0.9 A §
(n=3)* 167.6 ± 77.2 Means of each type of comparison followed by distinct letters differ statistically (p<0.05).
Means of each type of comparison, which are not followed by letters, do not differ from each other. AUC: Area under the curve of pH fall; cH+: area of hidrogenionic concentration
§: data transformed by log10; #: data transformed by 1/AUC.
*The n value is different due to the exclusion of outliers from genotype 13su (values of 4.22 for final pH and 0.10 for cH+ area after 15 min)
DISCUSSION
Regarding the data of genotypic diversity, we found that most of the volunteers harbored just one genotype in saliva, which was also identified in the biofilms (2, 15) (Table 1). Even though some studies discuss that saliva does not carry all genotypes present in the oral cavity (27), there are evidences that a positive correlation between mutans streptococci in saliva and those present in dental biofilm exists (31) and we believe that saliva reflects, at least, those genotypes present at higher proportions in teeth biofilms as previously reported by some studies (2, 15) and also observed in the present research.
Our data suggest, in addition, that there were no expressive differences in genotypic diversity among biofilms formed either in the presence or absence of sucrose. In this context, some studies have suggested that frequent exposure to sucrose and even the frequent pH perturbations may be related to an increased genotypic diversity of microorganisms in oral cavity (1, 24). However, it is difficult to correlate our data with these previous one since the experimental conditions evaluated in these studies were different compared to ours. Nevertheless, despite the absence of expressive differences in genotypic diversity, the distinct conditions of biofilm formation allowed that specific genotypes were also isolated from biofilms, although in a lower proportion (Table 1), as previously described (2). These particular genotypes might be present in saliva below the detection limit of the microbiological method used (27), and these specific conditions may have enhanced their proportions in biofilms.
In relation to the phenotypic traits, genotypes isolated from biofilms formed in the presence of sucrose seemed to be more acid tolerant, since they showed higher ratio of counts of viable cells at pH 2.8 T30 than those isolated from biofilms formed in the absence of this carbohydrate (Table 2). Moreover, those genotypes exclusively isolated from biofilms formed in the presence of sucrose showed higher ratio of counts of viable cells at pH 5.0 T60 (Table 2). Also, genotypes exclusively isolated from biofilms formed in the presence of sucrose (7su, 10su, 11su and
13su) were more acidogenic (in terms of cH+ area after 15 min and final pH) than those exclusively isolated in biofilms not exposed to sucrose (4ddw, 6ddw, 9ddw, 15ddw, 16ddw, 17ddw, 18ddw and 19 ddw) (Table 3). With the exception of data of
cH+ area (after 15 min), which means how fast the biofilm pH decreased due to
acid production, the other statistical significant differences showed above might be seen with care as they may not be clinically relevant either considering that the pH drop in oral cavity due to carbohydrate fermentation may not reach values as low as pH 2.8 and the relevance of a higher cell viability after 60 min at pH 5.0 or even considering the final pH evaluation, since in an in vitro study all genotypes had chance to decrease the pH and probably the first minutes were the most important to observe. Nevertheless, these findings suggest that genotypes isolated from biofilms formed in the presence of sucrose may be more virulent. Probably, these genotypes might have developed an adaptive response to the frequent pH fall due to sucrose exposure, which may have increased their acid-tolerance. In addition, the higher acidogenicity of genotypes exclusively isolated in the presence of sucrose may suggest that not only the modifications induced by microorganisms in biofilms, but also their virulence traits, are more important than only their relative numbers in biofilms (9). Moreover, as the expression and modulation of virulence factors related to caries development are dependent on environmental conditions (7), this may help explain the higher cariogenicity and acidogenicity of biofilms formed in the presence of sucrose (8).
Since the F-ATPase pump activity has an inverse relationship with the environmental pH (3), we hypothesized that genotypes isolated from biofilm formed under frequent pH fall due to sucrose exposure may show higher activity than those isolated from biofilms not exposed to this carbohydrate. However, in our experimental conditions, the difference did not reach statistical significance (p=0.06) (Table 2). We may consider that the absence of difference in F-ATPase activity between these two distinct conditions can be due to some inherent problems regarding the protocol adopted to evaluate the activity of this pump. It has been suggested that the toluene, used during the cell permeabilization step,
may inhibit F-ATPase activity (30). Additionally, the authors discussed that toluene may induce ruptures in the cell membrane causing an outflow of the cytoplasm ATPases. In this case, the values of F-ATPase activity may be a result of not only the membrane-bound F-ATPase activity but also the non-specific activity of intracellular ATPases, which might have interfered with our results, eliminating any differences among genotypes. Thus, it would be interesting in future studies to evaluate a modified protocol, considering these observations.
Besides F-ATPase acitivity, other mechanisms might be involved in acid-tolerance. In this context, under an acidic stress condition, there is a shift in the membrane fatty acid profile of S. mutans, which may decrease its permeability to protons (10). In addition, the modulated expression of some proteins and an increased expression of DNA repair enzymes under low pH might be also involved in acid tolerance of S. mutans (32, 11). As these mechanisms also play a role in the acid tolerance of S. mutans, it would be relevant to study them in future studies in order to clarify their roles in the aciduricity of different S. mutans genotypes.
Furthermore, it is shown that distinct genotypes may present distinct phenotypic traits. In the present study, distinct genotypes showed distinct acid tolerances, considering not only the counts of viable cells after 30 min at pH 2.8 or
the F-ATPase activity, but also distinct acidogenicities, regarding AUC and cH+
data, within 5 of 6 volunteers (by Kruskal-Walis test; p<0.05; data not shown). These findings are in agreement with previous data which showed that distinct genotypes may show distinct ability to sinthesize insoluble extracellular polysaccharides, different activities of glucosyltransferases or even different cariogenic potential in an animal caries model (12, 20, 22). Perhaps, an evaluation of these phenotypic traits in an in vitro biofilm model, additionally, might also better reflect the behavior of the genotypes in a stress condition induced by the frequent pH fall due to sucrose exposure.
Overall, the findings of the present study suggest that, even though there was not a considerable difference in the genotypic diversity between biofilms
formed either in the presence or absence of sucrose, biofilms formed under a high cariogenic condition harbored more aciduric and acidogenic genotypes.
ACKNOWLEDGMENTS
The study was supported by FAPESP (process # 03/10972-8; 05/03089-6 and 07/08000-9). The authors acknowledge the volunteers for their valuable participation.
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CAPÍTULO 2
Phenotypic analysis of S. mutans genotypes in planktonic and biofilm conditions”
R.A. Arthur, R.A Ccahuana-Vásquez, P.L. Rosalen, R.O. Mattos-Graner, A.A. Del Bel Cury, J.A. Cury, C.P.M. Tabchoury
Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil
Short title: S. mutans phenotypic traits as planktonic cells and biofilm
Corresponding author:
Cínthia Pereira Machado Tabchoury Av. Limeira, 901 13414-903, Piracicaba, SP Brazil Phone: #55-19-21065304 Fax: #55-19-21065212 E-mail: cinthia@fop.unicamp.br
ABSTRACT
S. mutans genotypes may show distinct phenotypic traits; however little is
known about the phenotypic traits of S. mutans genotypes isolated from an in situ dental biofilm formed in the presence or absence of a high cariogenic challenge. Thus, this study aimed to evaluate the aciduricity and acidogenicity of these genotypes grown in a planktonic condition and also their enamel demineralization potential in an in vitro biofilm model. Sixteen genotypes from 3-day in situ biofilm, formed in the presence or absence of sucrose exposure, were evaluated regarding acid susceptibility, F-ATPase activity and ability to lower the pH through glycolysis in planktonic conditions. Eight of these genotypes, with the highest or the lowest aciduricity and acidogenicity, were grown during 5 days as an in vitro biofilm exposed 8x/day to 10% sucrose solution and the acidogenicity, the ability to synthesize extracellular polysaccharides and the enamel demineralization potential of these genotypes were also evaluated. Distinct genotypes showed distinct F-ATPase activity and distinct acidogenicities, although the difference in the acidogenicity was less pronounced. In addition, genotypes isolated from biofilms formed in the presence of sucrose might be more acid-tolerant in planktonic conditions than those isolated in sucrose absence. In biofilm conditions, the genotypes were not different regarding either their ability to produce EPS or their acidogenicities, but, they showed distinct enamel demineralization potential. Either in planktonic or biofilm conditions, distinct genotypes show distinct phenotypic traits.
